Solid-State Nanopore Single-Molecule Sensing of DNAzyme

Subscriber access provided by UNIVERSITY OF LEEDS

Functional Nanostructured Materials (including low-D carbon)

Solid-State Nanopore Single-Molecule Sensing of DNAzyme Cleavage Reaction Assisted with Nucleic Acid Nanostructure Libo Zhu, Ying Xu, Irshad Ali, Liping Liu, Hongwen Wu, Zuhong Lu, and Quanjun Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09505 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Solid-State Nanopore Single-Molecule Sensing of DNAzyme Cleavage Reaction Assisted with Nucleic Acid Nanostructure Libo Zhu1, Ying Xu1, Irshad Ali1, Liping Liu1,2, Hongwen Wu1,3, Zuhong Lu1, Quanjun Liu1* 1. State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, No. 2, Sipailou, Nanjing 210096, People’s Republic of China. 2. Guizhou Institute of Technology, Guizhou, Guiyang, 550003, People’s Republic of China. 3. Department of Medical Devices, First Affiliated Hospital of Nanchang University, Nanchang, 330006, China. Corresponding author: [email protected]

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The detection and investigation of biomolecules at a single molecule level is important for improving diagnosis in biomedicine. Solid-state nanopores are a unique tool that have the potential to accomplish this task, because they are label-free, and require only low sample consumption. However, the event-readouts of current small polymer molecules are still limited due to its relatively large size and low signal-to-noise ratios. Here, we present a rapid sensing approach for the detection of GR-5 DNAzyme cleaving specific substrate reactions using relatively larger size silicon nitride nanopores by introducing a type of nucleic acid nanostructure (DNA tetrahedron) as a carrier. The proposed method is convenient and sensitive enough to detect the cleavage reactions by identifying translocation events before and after reactions with nanomolar concentrations of the target sample. Furthermore, this assay was also carried out by using larger size nanopores (60 nm diameter) to achieve the DNAzyme cleavage sensing with the same sample concentration. This approach can improve event detectability of other smaller molecules’ translocation, which opens up a wide range of applications for analytes detection by incorporating solid-state nanopores. Nucleic acid nanostructure-assisted nanopore sensing can promote the development of single molecule studies. KEY WORDS: DNAzyme, Silicon nitride Nanopore, Sensing, Single-molecule, Nucleic acid Nanostructure.

2


Page 2 of 32

Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Many techniques in medicine and biotechnology are based on nucleic acid hybridization. Due to their tunable properties and functions, there is an increasing interest in investigating nucleic acid hybridization and various hybridized structures.1-3 Functional nucleic acids consist of DNAzymes and aptamers and are a type of nucleic acid strand with a catalytic effect when in the presence of cofactors such as specific inorganic, organic or biomolecules.4 DNAzyme as a kind of functional DNA can cleave substrates after the addition of specific metal ions.5 As a result of its high metal ion specificity, DNAzyme is frequently used as a metal ions sensing platform. There are various methods available to detect DNAzyme cleavage reactions, such as electrochemical,6-7 colorimetric,8-9 and fluorescence methods.10-11 Although these traditional techniques can detect the cleavage reaction sensitively and selectively, these techniques have some drawbacks: they are low- efficiency, time-consuming, and they require complex systems. For example, most of the electrochemical methods mainly involve immobilizing the molecules on metal electrodes and they also depend on the direct oxidation detection of the molecules, immobilized on metal electrodes. Because of the selectivity and sensitivity of the metal electrode, the system can not guarantee the optimal detection conditions. At the same time, there is possibility of electrode contamination by this method. Complication in colorimetric and fluorescence methods, involve the requirements of high

3



enzyme extraction and reaction terminated. For example, fluorescence detection requires longer detection time and expensive reagent consumption. In view of this, there is a need to develop a kind of simple, label-free and low time-consumption measurement technique to detect the presence of DNAzyme cleavage reactions, in order to overcome the abovementioned disadvantages. Nanopores are an increasingly popular single-molecule sensing approach that can detect individual molecules in a label-free fashion.12-14 A nanopore consists of a nanoscale channel on a biological15 or artificial film.1, 16 Target molecules pass through or interact with this nanoscale channel, and changes in the open-pore ionic current can be monitored. Its inherent advantages such as high temporal-spatial resolution,17 ultrasensitivity make it a unique single-molecule sensor.18 Especially, solid-state nanopores, its pore size and shape is controllable, high physical and chemical stability and surface modification ability.19 Because of these advantages, nanopores have become an important tool in detecting various molecules, such as, nucleotides,20-23 peptides24-27 and protein,28-31 protein denaturation32-34 and enzymatic reactions.35-37 Therefore, biological nanopores are a type of nanopore sensor, and owing to the small diameter of the sensing-area (less than 3 nm), they have the ability to detect the single strand DNAzyme cleavage products.38-40 Nevertheless, there are two drawbacks of biological nanopores that must be taken into account. One issue is the stability of the lipid bilayers that are disrupted by protein insertion, and the other is the low reaction condition

4


Page 4 of 32

Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tolerance, which limits its applications. Solid-state nanopores are usually fabricated on an artificial membrane, making them highly stable and efficient under extreme conditions. However, to date, no studies have examined DNAzyme detection using solid-state nanopores with its low signal-to-noise ratios (S/N). Fortunately, the inclusion of modified DNA carriers can improve the signal-to-noise ratio of nanopores.41 Introduction of this technique can help us to enhance the sensitivity and event detection. In this paper, we propose a novel nucleic acid nanostructure based strategy to detect DNAzyme cleavage reactions by using solid-state nanopores. We utilized silicon nitride nanopores of different diameters (40 nm, 60 nm) to sense GR-5 DNAzyme cleavage reactions. To address the nanopore size limitations, introducing nucleic acid nanostructure can cause larger current blockages42 because of the 3D structure of the nucleic acid nanostructure. We designed and synthesized nucleic acid nanostructures (DNA tetrahedrons) that have strong affinity towards target DNAzyme molecule. Owing to their highly predictable geometric structure, programmable design, and highly precise dimensions, nucleic acid nanostructures have been used to carry out numerous functional application.43-45 In our experiment, the edge length of the DNA tetrahedron is ~9 nm. The DNA tetrahedron-binding

DNAzyme

(TBD)

complex

provides

an

improved

signal-to-noise ratio due to an increase in excluded volume, induced by the particularity of the event because of its distinctive spatial structure and carried charge. On this basis, different translocation events that are produced by the TBD complex and its cleavage

5



products have been monitored. Furthermore, single-molecule DNAzyme cleavage reactions can be observed in real-time by incorporating the presented methodology to detect translocation events using different diameters of nanopores.

EXPERIMENTAL SECTION Chemicals and Apparatus Sodium chloride (NaCl), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were provided by Di Bo Chemical Technology Co., Ltd (Shanghai, China). The 1×TE Buffer (10 nM Tris, 1 mM EDTA, pH 8.0) was purchased from KeyGEN BioTECH Co., Ltd. (Nanjing, China), and the 1×TM Buffer (20 mM Tris, 50 mM MgCl2, pH 7.5) was purchased from Kang Lang Bio Technology Co., Ltd. (Shanghai, China). Additionally, 2-(N-morpholino)ethanesulfonic acid hydrate (MES Buffer, pH 5.5) was provided from Biotopped Co., Ltd. (Beijing, China). Lead(II) nitrate (Pb(NO3)2) was provided by Sinopharm Chemical Reagent Beijing Co., Ltd. The DNA sequences for DNA tetrahedron synthesis were provided and purified by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Reagents were prepared using deionized water from a Milli-Q water purification system (resistivity of 18.2 MΩ/cm, 25 °C, Millipore Corporation, Billerica, MA, USA) and was filtered through 0.02 µm in a FEI Strata 201 FIB system (FEI Co., Hillsboro, OR, USA). A Zetasizer (Malvern Zetasizer Nano ZS), Scanning Electron Microscopy (SEM) Axiostar plus (ZEISS Axiostar plus), an FEI Titan 80-300 Transmission Electron Microscope (TEM) system (FEI Co., Hillsboro, OR, USA)

6


Page 6 of 32

Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and an Axopatch 700B (Molecular Devices, Inc., Sunnyvale, CA, USA) were used. Silicon Nitride Nanopores Fabrication and Measurement The size of the nanopore chips were 3 × 3 mm2, with a 100-nm-thick Si3N4 freestanding membrane supported by a silicon wafer. This Si3N4 membrane was deposited by Low Pressure Chemical Vapor deposition (LPCVD) with a 500 × 500 µm2 square open window exposed for KOH wet etching. Nanopores were drilled by bombarding the chip with Ga+ ions using an FEI Strata 201 FIB system (FEI Co., Hillsboro, OR, USA) at an acceleration potential of 30 kV. After fabrication, nanopore chips were cleaned using Piranha solution at 80 °C for 30 min, rinsed with deionized water, and finally dried under nitrogen (N2). Next, the nanopore chip was mounted in a custom-built Teflon fluidic cell, and two Viton O-rings were used to separate the two sides of the chip, forming two reservoirs to ensure the only path for the ionic current was through the nanopore channel. Experiments were performed using 0.5 M NaCl (1×TM buffer, pH 7.5). Two reservoirs were connected to the patch clamp amplifier by two Ag/AgCl electrodes. The ionic current was recorded by 700B patch clamp amplifier, and it was filtered at 10 kHz by a low-pass filter. The whole instrument was placed in a double Faraday cage enclosure. Before the experiments, open-pore conductance of the nanopores (G0) was measured and compared against the measurements of the SEM to confirm the diameters of the nanopores. Then, samples were added to the cis side, and a positive biased voltage was

7



applied. Under the applied biased voltages, the target molecules were driven through the nanopore by electrophoretic force, which resulted in different translocation events. All of the nanopore data were recorded and extracted using Clampfit. Sample Preparation The DNA tetrahedron synthesis samples (ssDNA1, ssDNA2, ssDNA3 and ssDNA4 oligonucleotides), DNAzymes and substrate strands samples in dried form, were dissolved in 1×TE Buffer (10 nM Tris, 1 mM EDTA, pH 8.0) to obtain stock solutions of 100 µM, respectively. The stock solutions were stored at −20 °C. Synthesis of DNA Tetrahedrons The samples of ssDNA1, ssDNA2, ssDNA3 and ssDNA4 were dissolved in 1×TM buffer (20 mM Tris, 50 mM MgCl2, pH 7.5), and then, the mixtures were heated to 95 °C for 10 min using a PCR instrument, followed by rapid cooling to 4 °C. Finally, the mixtures were placed at 56 °C for 1 h.

RESULTS and DISCUSSION Design and Synthesis of DNA Tetrahedron-binding DNAzyme Complexes To enable detection of DNAzyme cleavage reactions with the silicon nitride (Si3N4) nanopore, we aimed to identify whether the translocation events before and after the reaction would be different. Schematic concepts of detecting DNAzyme cleavage reactions using Si3N4 nanopore are shown in Figure 1a.

8


Page 8 of 32

Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Schematic of nanopore detection of DNAzyme cleavage reactions. (a) Illustration of a silicon nitride nanopore sensing GR-5 DNAzyme cleavage reactions based on DNA tetrahedrons. Insert: 1, DNA tetrahedron-binding DNAzyme (TBD) complexes translocation events; 2, TBD complexes cleavage products translocation events; 3, the cleaved substrate strands translocation events. (b) All of DNA sequences used are listed: ssDNA1, ssDNA2, ssDNA3 and ssDNA4 were used to synthesized the DNA tetrahedron, and an amino group (-NH2) was modified at the 5’ terminal of ssDNA3. A carboxyl group (-COOH) was modified at the 5’ terminal of the GR-5 DNAzyme. Substrate: the sequence of the GR-5 DNAzyme specified cleavage substrate strand. (c) Current– Voltage (I–V) curve in the range −200 to 200 mV for one of silicon nitride nanopores used in this study. Insert: scanning electron microscopy (SEM) image of a nanopore (scale bar is 50 nm).

Recently, Fan and coworkers reported that nucleic acid nanostructures of various shapes were synthesized successfully46-48. Due to inherent advantages such as high 9



predictable geometric structure and dimensions, the nucleic acid nanostructures had a wide applications, such as, electrochemical biosensing,49-51 optical sensing,52 and microfluidic technology.53 Inspired by their work, the designed nucleic acid sequences were utilized (Figure 1b) to build nucleic acid tetrahedrons. Each edge of the tetrahedron contains 26 base pairs, and every edge is ~9 nm in length. To confirm that the DNA tetrahedrons were synthesized correctly, 2% agarose gel electrophoresis assays were performed. As demonstrated in Figure 2a, mobility of the tetrahedron was lower than the other hybridized ssDNA strands. In addition, the hydrodynamic size of the tetrahedron was measured by dynamic light scattering (DLS), showing the average measurement diameters was ~10.3±2.53 nm, as demonstrated in Figure 2b. Figure 2c shows the TEM images of synthesized DNA tetrahedrons. After confirming the DNA tetrahedron size, the hybridization experiments between GR-5 DNAzyme and its specified substrate strands were performed. The secondary structure of the GR-5 DNAzyme hybridized with the specific substrate strand is shown in Figure S1 (Supporting information S1). The samples of GR-5 DNAzymes and substrate strands were mixed and incubated at room temperature for 60 min in 1×TM buffer (pH 7.5).54 The final concentrations of the hybridization products were 5 nM. Next, we bound the single DNA tetrahedron to individual strands of the hybridization product by means of an EDC and NHS cross-linked system. The carboxyl groups can be activated by EDC, forming an amine-reactive o-acylisourea intermediate, and the NHS stabilizes the intermediate compound by converting it into an

10


Page 10 of 32

Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amine-reactive NHS ester. The amine-reactive NHS esters react with ethylenediamine to yield amide bonds.55 Therefore, a carboxyl group (-COOH) at the 5’ terminus of every DNAzyme strand can cross-link with an amino group carried by the DNA tetrahedron. Upon completion of the DNA tetrahedron-binding DNAzyme complex synthesis, we validated the specificity of the substrate’s cleavage ability in the presence of lead(II) by a colorimetric method, the experimental details are shown in S2 (Supporting information S2). For the detection and analysis of the cleavage reaction, silicon nitride nanopores of 40 nm were obtained, and their diameters were measured by SEM imaging and characterized by typical current–voltage (I-V) curves (Figure 1c). The test of stability for the nanopore is shown in Figure S3 (Supporting information S3). As described by Wanunu and coworkers,56 according to the current–voltage (I-V) curves, the calculated value of the nanopore diameter is ~40 nm in 0.5 M NaCl solution.

11



Figure 2. Characterization of DNA tetrahedrons. (a) 2% Agarose gel electrophoresis image of the formation of the DNA tetrahedron. Lane 1, DNA marker; Lane 2 and Lane 3, one strand of ssDNA; Lane 4 and Lane 5, hybridization of two strands of ssDNA; Lane 6 and Lane 7, hybridization of three strands of ssDNA; Lane 8, hybridization of the DNA tetrahedron. (b) Dynamic Light Scattering (DLS) distribution of the hydrodynamic size of DNA tetrahedrons. (c) TEM images of the synthesized DNA tetrahedrons.

Single Molecule Detection of TBD Complexes To demonstrate the feasibility of detecting GR-5 DNAzyme cleavage by using relatively larger size nanopores, we aimed to distinguish the translocation signals of the DNA tetrahedron-binding DNAzyme (TBD) complex and its cleavage products. Therefore,

12


Page 12 of 32

Page 13 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TBD complex transport experiments were carried out in 0.5 M NaCl solution firstly. The nanopore chip was mounted into a custom fluidic cell, and an electrolyte solution (0.5 M NaCl dissolved in 1×TM buffer) was then used to fill the cell. Subsequently, the samples of TBD complexes at final concentrations of 5 nM were added into the cis reservoir of the fluidic cell and corresponding positive voltages across the membrane were applied. As expected, ionic current transient changes through the nanopore were observed. To quantify the changes of ionic current, statistical analysis for current blockage (∆I) and dwell time (∆t) of translocation events under varying biased voltages were performed. We analyzed the current blockage (∆I1) and dwell time (∆t1) for TBD complexes translocation. The ∆I1 histograms were fitted by Gaussian function with a mean current change of 167.8±3.91, 224.6±6.01and 291.9±4.31 pA at 500, 700 and 900 mV, as shown in Figure 3a-c. The histograms of current blockage at 600 and 800 mV are shown in Figure S4a-b (Supporting information S4). Figure 3d-f depicts the histograms of dwell time; the mean values of dwell time were 14.9±4.01, 7.3±3.71 and 3.6±1.01 ms, respectively, by Gaussian fitting. Other voltage conditions are shown in Figure S5a-b (Supporting information S5). As described above, the changes in ∆I1 and ∆t1 by holding different voltages shows that these are translocation events rather than collisions. The relationship of both quantities with the biased voltage was analyzed. Figure 3g-h shows changes in current blockage and dwell time versus biased voltage. The current blockages are linearly dependent on biased voltage; the dwell time changes are exponentially

13



dependent on biased voltage in the range 500–900 mV, suggesting that the TBD complexes translocation is a voltage-activated process. In addition, a two-dimensional scatter plot of (∆I1) versus (∆t1) of all events is shown in Figure 3i. The difference in events under varying voltages can be observed clearly from the different clusters of events.

Figure 3. Statistical analysis of DNA tetrahedron-binding DNAzyme (TBD) complexes’ translocation events. (a)–(c) Current blockage (∆I1) histograms for TBD complexes transported through a 40-nmdiameter Si3N4 nanopore at 500, 700 and 900 mV. (d)–(f) Dwell time histograms for TBD complexes translocation at 500, 700 and 900 mV. (g) The current blockage (∆I1) of a translocation event as a function of biased voltage. (h) The dwell time (∆t1) of a translocation event as a function of biased voltage. (i) Scatter plot of current blockage (∆I1) versus dwell time (∆t1) under different voltages.

14


Page 14 of 32

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Single Molecule Detection of DNAzyme Cleavage Reaction Further experiments were conducted to test the ability of sensing DNAzyme cleavage reactions. Recently, some groups have reported that electrochemical techniques can be used to detect DNAzyme cleavage reactions.57-58 Lead (II) (Pb2+) dependent GR-5 DNAzyme can specifically cleave the complementary substrate strand at the ribonucleotide site (rA) of the substrate. Based on this reaction principle, we added Pb2+ (final concentration of 100 nM) into the TBD complexes solution, and incubated it in the reaction buffer (0.5 M NaCl dissolved in 1×TM buffer) at room temperature for 1 hour. Afterwards, the cleavage products solution was added into the cleaned cis reservoir of a fluidic cell and corresponding biased voltages were applied. It is interesting to note that a new type of event was observed; the current blockage (∆I2) and dwell time (∆t2) are different from that of the TBD complexes’ translocation events. Figure 4a-c shows the distribution of current blockage (∆I2). The histograms were fitted by a Gaussian function with a mean current change of 92.6±13.61, 131.9±2.67 and 239.5±5.38 pA at 500, 700 and 900 mV, respectively. The histograms of current blockage at 600 and 800 mV are shown in Figure S6a-b (Supporting information S6). The histograms of dwell time (∆t2) are shown in Figure 4d-f, where the mean values of ∆t2 were 7.6±2.84, 5.2±2.55 and 3.1±1.32 ms. Other voltage conditions are shown in Figure S7a-b (Supporting information S7). Based on the differences in current blockage and dwell time, these differences can be ascribed to translocation of different molecules, and

15



according to conventional nanopore sensing principles,59-62 we speculate there are smaller sized molecules passed through the nanopore. A scatter plot of current blockage (∆I2) versus dwell time (∆t2) is shown in Figure S8 (Supporting information S8). To confirm that the new type of event was not produced by collision, we also analyzed the dependence of current blockage and dwell time on biased voltages, as shown in Figure S9 (Supporting information S9), and as expected, ∆I2 and ∆t2 were dependent on biased voltage.

Figure 4. Statistical analysis of TBD complexes cleavage products translocation events. (a)–(c) Current blockage (∆I2) histograms for the cleavage products transport through a 40-nm-diameter Si3N4 nanopore at 500, 700 and 900 mV. (d)–(f) Dwell time histograms for the cleavage products at 500, 700 and 900 mV.

As previously reported,58 the products of GR-5 DNAzyme cleavage end up in three parts: the DNAzyme strand and two short cleaved strands of substrate. However, the only difference in the cleavage products is that the DNAzyme strand carried the DNA 16


Page 16 of 32

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tetrahedron in our experiments, as displayed in Figure 1a. To ensure that the new type of translocation events were caused by translocation of the cleavage products, control experiments were performed with designed products. We directly synthesized the DNA tetrahedron and combined it with the DNAzyme strand; the component and structure were the same as the cleavage products (DNA tetrahedron carrying the DNAzyme strand). The designed products (final concentration 5 nM) were injected into the fluidic cell of the nanopore and biased voltages (500, 600, 700, 800 and 900 mV) across the nanopore membrane were applied. The schematic concept of the silicon nitride nanopore detecting the designed products is described in Figure 5a. Figure 5c-e shows the histograms that were fitted by a Gaussian function with a mean current blockage (∆I2c) of 86.3±6.39, 138.1±4.88 and 238.6±1.87 pA (500, 700 and 900 mV) for the designed products translocation, respectively. The mean values of the current blockage (∆I2c) were almost same as the mean values of ∆I2. The fractional current blockage (∆I/I0) versus the voltage data fit well to the Gaussian function (Figure 5f); the values of ∆I/I0 were 0.0067, 0.0069 and 0.0072 for TBD complexes cleavage products (red), and 0.0062, 0.0072 and 0.0071 for designed products (black). Based on these values, ∆I/I0 remained independent of biased voltage and almost maintained a constant value.

17



Figure 5. (a) Schematics of the designed products (DNA tetrahedron carrying the DNAzyme strand) translocating through a Si3N4 nanopore. Insert: representative current traces. (b) Schematics of the GR-5 DNAzyme specified cleavage substrate strands translocating through the same nanopore, and no translocation events appeared. (c)–(e) Current blockage (∆I2c) histograms for the designed products passing through the nanopore at 500, 700 and 900 mV. (f) Mean fractional current blockage (∆I/I0) versus biased voltage for TBD complexes cleavage products’ translocation events (red) and designed 18


Page 18 of 32

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


products’ translocation events (black). (g) The current blockage (∆I2c) of a translocation event as a function of biased voltage, which shows a linear increase of current blockage in response to an increased voltage. (h) The dwell time (∆t2c) of the translocation event as a function of biased voltage, which shows an exponential increase dependent on the biased voltage. (i)–(k) Scatter plot of current blockage (∆I) versus dwell time (∆t) for TBD complexes’ cleavage products’ translocations (red) and designed products’ translocations (black). The corresponding distributions of dwell times (∆t) are shown above the scatter plots.

Moreover, we detected the DNAzyme substrate strands using the same nanopore. The sample of corresponding substrate strands was added into the cleaned cis reservoir and biased voltages were applied; however, it showed no detectable events (Figure 5b). According to previous research,62 by employing a significantly larger nanopore than the size of the analyte and a low signal-to-noise ratio of the solid–state nanopore, we failed to identify the substrate strand translocation events. In addition, the current blockage (∆I2c) and dwell time (∆t2c) are dependent on biased voltages, as shown in Figure 5g, h. In addition, the scatter plot of the current blockage versus dwell time clearly demonstrates two populations that are almost coincident, respectively, at different voltages, indicating that the translocation events were produced by almost the same analytes. The corresponding distributions of dwell times are shown on the top of the scatter plots (Figure 5i-k). On the basis of the above results, it can be validated that the new type of events were

19



only produced by the population of the cleavage products (DNA tetrahedron carrying the DNAzyme strand). In a continuation of the above studies, structural features of different analytes were analyzed. We noted that current blockages (∆I1) were larger than current blockages (∆I2) and the dwell times (∆t1) were longer than ∆t2. To distinguish molecular translocation events, the specific shapes within each ionic current signature were further analyzed. Figure 6a and 6b show a continuous current versus time traces from a 40-nm-diameter nanopore drilled in a 100-nm-thick Si3N4 membrane measured at different voltages (500, 700 and 900 mV). It can be seen that the frequency of translocation was also a voltage-activated process, and the translocation frequency increases with increasing voltage. A schematic concept of silicon nitride nanopore detection corresponding to the analyte and representative current traces of each type of analyte transport are shown to the left, respectively. It can also be noted that the TBD complexes’ translocation events have deeper blockage magnitudes by observing the continuous current traces. To quantify the difference in the TBD complexes and the cleavage products’ translocation, the fractional current blockage (∆I/I0) was investigated. Figure 6c shows the distribution of two-dimensional ∆I/I0 for the TBD complexes’ transport at different voltages, it should be noted that the main part of the ∆I/I0 distributions appear similar, and the corresponding Gaussian fitting value is 0.011±0.0079. The Gaussian fitting value of ∆I/I0 for the cleavage products translocation is 0.007±0.0008, as shown in Figure 6e. Moreover, we

20


Page 20 of 32

Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


observed that the values of (∆I/I0) remained independent of voltage, shown in Figure 6d and 6f, respectively. The dwell time (∆t) versus biased voltage is shown in Figure S10 (Supporting information S10). In general, our experimental results showed that the DNAzyme cleavage reactions could be detected using a relatively larger Si3N4 nanopore via distinguishing different translocation events before and after the reactions.

Figure 6. Discrimination of the different analytes’ translocation events. Continuous current versus time for TBD complexes’ translocations (a) and the cleavage products’ translocations (b). Schematic of the concept of sensing corresponding analyte and representative current traces are shown to the left respectively. (c) The histogram of normalized for TBD complexes translocation events. (d) Mean fractional current blockages (∆I1/I0) as a function of applied biased voltage. (e) The histogram of

21



normalized for cleavage products translocation events. (f) Mean fractional current blockages (∆I2/I0) as a function of applied biased voltage.

Detection of DNAzyme Cleavage Reaction using Different Diameter Nanopores After demonstrating the ability to detect the cleavage reaction by using a 40-nm-diameter silicon nitride nanopore, we also conducted the same experiments using a 60-nm-diameter nanopore.

Figure 7. Statistical analysis of translocation events across a 60 nm diameter nanopore. (a) Current– voltage (I–V) curve in the range −200 to 200 mV for a silicon nitride nanopore with diameter ~60 nm.

22


Page 22 of 32

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Insert: SEM image of a nanopore (scale bar is 50 nm). (b) Scatter plot of current blockage versus dwell time. (c)–(e) Current blockage (∆I) histograms. (f)–(h) Dwell time (∆t) histograms. (TBD complexes (blue) and their cleavage products (green)).

A typical current–voltage (I–V) curves and SEM imaging are shown in Figure 7a. A scatter plot of current blockage versus dwell time at different voltages is displayed in Figure 7b. It clearly demonstrates two clusters of populations for different analytes’ translocation, indicating that the translocation events of the TBD complexes and the cleavage products can be distinguished. The histograms of current blockage were fitted by a Gaussian function, as shown in Figure 7c-e. The mean values of current blockage (∆I1’) for the TBD complexes translocations were 93.1, 135.8 and 223.9 pA; the current blockage (∆I2’) for the cleavage products translocations were 74.9, 103.5 and 188.2 pA. Figure 7f-h shows the histograms of dwell time (∆t1’ and ∆t2’) and the corresponding fit mean values ∆t1’ were 13.9, 6.9 and 3.3 ms and for ∆t2’ were 6.7, 5.0 and 1.5 ms, respectively. Continuous current versus time traces for a 60-nm-diameter nanopore at 700 mV are shown in Figure 8a, with the representative current traces shown on the right.

23



Figure 8. (a) Continuous current versus time for TBD complexes (top) translocations and the cleavage products (bottom). Representative current traces are shown on the right. (b) Mean fractional current blockage (∆I/I0) versus biased voltage for TBD complexes’ translocation events (red/blue) and cleavage products’ translocation events (black/green). (c) The dwell time (∆t) changes versus biased voltage for different diameter nanopore translocation events.

Finally, to compare translocation events of different diameter nanopores, we analyzed the fractional current blockage (∆I/I0) versus voltage data (500, 700, 900 mV) (Figure 8b). This suggests that the value of ∆I/I0 are independent of voltage. However, it decreased with an increase in the diameter of the nanopore. The dwell time (∆t) changes versus the biased voltage for different diameter nanopores’ translocation events is shown in Figure 8c. 24


Page 24 of 32

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS The ability to detect DNAzyme cleavage reactions using solid-state nanopores has tremendous potential in medical diagnostic and molecule structure studies. In this work, we proposed a novel approach to detect GR-5 DNAzyme cleavage reactions using silicon nitride nanopores with relatively larger sizes. We designed and synthesized DNA tetrahedrons, and then, we combined them with GR-5 DNAzymes to construct complexes. Then, silicon nitride nanopores with ~40 nm diameters were used to sense DNAzyme cleavage. This approach was used to analyze the current blockage (∆I) and dwell time (∆t), which helped us to clearly detect and discriminate the difference in translocation

25



events before and after the reaction. Additionally, we also used 60-nm-diameter silicon nitride nanopores to test their sensing ability. Thus, using DNA tetrahedrons-based nanopores to detect DNAzyme cleavage reactions can enlarge the application range of conventional solid-state nanopore sensors, providing it is not necessary to fabricate ultra-small diameter nanopores for sensing small molecules.

AUTHOR INFORMATION Corresponding Author *Tel.: 025-83793283. *E-mail: [email protected]. ORCID: 0000-0002-9414-7892 Notes The authors declare no competing financial interest

ACKNOWLEDGEMENTS We acknowledge the financial support of the National Key R&D Program of China (2017YFC0906503), National Key R&D Program of China (2016YFA0501600), the Program of

26


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Shenzhen Knowledge Innovation (JCYJ20170817164740197).

REFERENCES (1) Singer, A.; Wanunu, M.; Morrison, W.; Kuhn, H.; Frank-Kamenetskii, M.; Meller, A. Nanopore based Sequence Specific Detection of Duplex DNA for Genomic Profiling. Nano letters 2010, 10 (2), 738-742. (2) Bell, N. A.; Keyser, U. F. Digitally Encoded DNA Nanostructures for Multiplexed, Single-molecule Protein Sensing with Nanopores. Nature nanotechnology 2016, 11 (7), 645-651. (3) Carson, S.; Wick, S. T.; Carr, P. A.; Wanunu, M.; Aguilar, C. A. Direct Analysis of Gene Synthesis Reactions Using Solid-State Nanopores. ACS nano 2015, 9 (12), 12417-12424. (4) Wang, Z.; Lee, J. H.; Lu, Y. Label‐Free Colorimetric Detection of Lead Ions with a Nanomolar Detection Limit and Tunable Dynamic Range by Using Gold Nanoparticles and DNAzyme. Advanced Materials 2008, 20 (17), 3263-3267. (5) Liu, G.; Zhang, L.; Dong, D.; Liu, Y.; Li, J. A Label-free DNAzyme-based Nanopore Biosensor for Highly Sensitive and Selective Lead Ion Detection. Analytical Methods 2016, 8 (39), 7040-7046. (6) Xiao, Y.; Rowe, A. A.; Plaxco, K. W. Electrochemical Detection of Parts-per-billion Lead via an Electrode-bound DNAzyme Assembly. Journal of the American Chemical Society 2007, 129 (2), 262-263. 27



(7) Tang, S.; Tong, P.; Li, H.; Tang, J.; Zhang, L. Ultrasensitive Electrochemical Detection of Pb2+ based on Rolling Circle Amplification and Quantum Dotstagging. Biosensors and Bioelectronics 2013, 42, 608-611. (8) Wei, H.; Li, B.; Li, J.; Dong, S.; Wang, E. DNAzyme-based Colorimetric Sensing of Lead (Pb2+) Using Unmodified Gold Nanoparticle Probes. Nanotechnology 2008, 19 (9), 095501-095505. (9) Jayabal, S.; Pandikumar, A.; Lim, H. N.; Ramaraj, R.; Sun, T.; Huang, N. M. A Gold Nanorod-based Localized Surface Plasmon Resonance Platform for the Detection of Environmentally Toxic Metal Ions. Analyst 2015, 140 (8), 2540-2555. (10) Qin, Y. C.; Gao, X. H.; Duan, L. H.; Fan, Y. C.; Yu, W. G.; Zhang, H. T.; Song, L. J. Effects on Adsorption Desulfurization of CeY ZeolitesAcid Catalysis and Competitive Adsorption. Acta Physico-Chimica Sinica 2014, 30 (3), 544-550(7). (11) Huang, P. J. J.; Liu, J.; Chem, A. Sensing Parts-per-Trillion Cd2+, Hg2+, and Pb2+ Collectively and Individually Using Phosphorothioate DNAzymes. Analytical chemistry 2014, 86 (12), 5999-6005. (12) Feng, J.; Liu, K.; Bulushev, R. D.; Khlybov, S.; Dumcenco, D.; Kis, A.; Radenovic, A. Identification of Single Nucleotides in MoS2 Nanopores. Nature nanotechnology 2015, 10 (12), 1070-1076. (13) Japrung, D.; Dogan, J.; Freedman, K. J.; Nadzeyka, A.; Bauerdick, S.; Albrecht, T.; Kim, M. J.; Jemth, P.; Edel, J. B. Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores. Analytical chemistry 2013, 85 (4), 2449-2456. (14) Yusko, E. C.; Bruhn, B. R.; Eggenberger, O. M.; Houghtaling, J.; Rollings, R. C.; Walsh, N. C.; Nandivada, S.; Pindrus, M.; Hall, A. R.; Sept, D. Real-time Shape Approximation and Fingerprinting of Single Proteins Using a Nanopore. Nature nanotechnology 2017, 12 (4), 360-367. (15) Cao, C.; Ying, Y.-L.; Hu, Z.-L.; Liao, D.-F.; Tian, H.; Long, Y.-T. Discrimination of Oligonucleotides of Different Lengths with a Wild-type Aerolysin Nanopore. Nature nanotechnology 2016, 11 (8), 713-718. (16) Kukwikila, M.; Howorka, S. Nanopore-based Electrical and Label-free Sensing of Enzyme Activity in Blood Serum. Analytical chemistry 2015, 87 (18), 9149-9154. (17) Ying, Y.-L.; Li, Z.-Y.; Hu, Z.-L.; Zhang, J.; Meng, F.-N.; Cao, C.; Long, Y.-T.; Tian, H. A Time-Resolved Single Molecular Train Based on Aerolysin Nanopore. Chem 2018. (18) Venkatesan, B. M.; Bashir, R. Nanopore Sensors for Nucleic Acid Analysis. Nature nanotechnology 2011, 6 (10), 615-624. (19) Wang, G.; Wang, L.; Han, Y.; Zhou, S.; Guan, X. Nanopore Stochastic Detection: Diversity, Sensitivity, and Beyond. Accounts of chemical research 2013, 46 (12), 2867-2877. (20) Cherf, G. M.; Lieberman, K. R.; Rashid, H.; Lam, C. E.; Karplus, K.; Akeson, M. Automated Forward and Reverse Ratcheting of DNA in a Nanopore at Five Angstrom Precision. Nature biotechnology 2012, 30 (4), 344-348. (21) Clarke, J.; Wu, H.-C.; Jayasinghe, L.; Patel, A.; Reid, S.; Bayley, H. Continuous Base Identification for Single-Molecule Nanopore DNA Sequencing. Nature nanotechnology 2009, 4 (4), 265-270. (22) Eid, J.; Fehr, A.; Gray, J.; Luong, K.; Lyle, J.; Otto, G.; Peluso, P.; Rank, D.; Baybayan, P.; Bettman, B. Real-time DNA Sequencing from Single Polymerase Molecules. Science 2009, 323 (5910), 133-138. (23) Cao, C.; Yu, J.; Wang, Y.-Q.; Ying, Y.-L.; Long, Y.-T. Driven Translocation of Polynucleotides through an Aerolysin Nanopore. Analytical chemistry 2016, 88 (10), 5046-5049. 28


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Piguet, F.; Ouldali, H.; Pastorizagallego, M.; Manivet, P.; Pelta, J.; Oukhaled, A. Identification of Single Amino Acid Differences in Uniformly Charged Homopolymeric Peptides with Aerolysin Nanopore. Nature Communications 2018, 9 (1), 966-978. (25) Huang, G.; Willems, K.; Soskine, M.; Wloka, C.; Maglia, G. Electro-osmotic Capture and Ionic Discrimination of Peptide and Protein Biomarkers with FraC Nanopores. Nature Communications 2017, 8 (1), 935-945. (26) Li, S.; Cao, C.; Yang, J.; Long, Y. T. Detection of Peptides with Different Charges and Lengths by Using the Aerolysin Nanopore. ChemElectroChem 2018. (27) Ying, Y.-L.; Yu, R.-J.; Hu, Y.-X.; Gao, R.; Long, Y.-T. Single Antibody–Antigen Interactions Monitored via Transient Ionic Current Recording Using Nanopore Sensors. Chemical Communications 2017, 53 (61), 8620-8623. (28) Balme, S.; Coulon, P. E.; Lepoitevin, M.; Charlot, B.; Yandrapalli, N.; Favard, C.; Muriaux, D.; Bechelany, M.; Janot, J. M. Influence of Adsorption on Proteins and Amyloid Detection by Silicon Nitride Nanopore. Langmuir the Acs Journal of Surfaces & Colloids 2016, 32 (35), 8916-8925. (29) Cressiot, B.; Oukhaled, A.; Patriarche, G.; Pastorizagallego, M.; Betton, J. M.; Auvray, L.; Muthukumar, M.; Bacri, L.; Pelta, J. Protein Transport Through a Narrow Solid-State Nanopore at High Voltage: Experiments and Theory. Acs Nano 2012, 6 (7), 6236-6243. (30) Rodriguezlarrea, D.; Bayley, H. Multistep Protein Unfolding During Nanopore Translocation. Nature nanotechnology 2013, 8 (4), 288-295. (31) Hu, Y.-X.; Ying, Y.-L.; Gu, Z.; Cao, C.; Yan, B.-Y.; Wang, H.-F.; Long, Y.-T. Single Molecule Study of Initial Structural Features on the Amyloidosis Process. Chemical Communications 2016, 52 (32), 5542-5545. (32) Freedman, K. J.; Jürgens, M.; Prabhu, A.; Ahn, C. W.; Jemth, P.; Edel, J. B.; Kim, M. J. Chemical, Thermal, and Electric Field Induced Unfolding of Single Protein Molecules Studied Using Nanopores. Analytical chemistry 2011, 83 (13), 5137-5144. (33) Japrung, D.; Dogan, J.; Freedman, K. J.; Nadzeyka, A.; Bauerdick, S.; Albrecht, T.; Kim, M. J.; Jemth, P.; Edel, J. B. Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores. Analytical chemistry 2013, 85 (4), 2449-2456. (34) Oukhaled, A.; Cressiot, B.; Bacri, L.; Pastorizagallego, M.; Betton, J. M.; Bourhis, E.; Jede, R.; Gierak, J.; Auvray, L.; Pelta, J. Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force. Acs Nano 2011, 5 (5), 3628-3638. (35) Zhu, L.; Gu, D.; Liu, Q. Hydrogen Peroxide Sensing Based on Inner Surfaces Modification of Solid-State Nanopore. Nanoscale Research Letters 2017, 12 (1), 422-431. (36) Chen, X.; G, M. R.; Ye, Z.; Zhang, Y.; Ma, R.; Xiang, J.; Guan, X. Label-free Detection of DNA Mutations by Nanopore Analysis. Acs Applied Materials & Interfaces 2018, 10 (14), 11519-11528. (37) Xi, D.; Li, Z.; Liu, L.; Ai, S.; Zhang, S. Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore. Analytical chemistry 2017, 90 (1), 1029-1034. (38) Yang, C.; Liu, L.; Zeng, T.; Yang, D.; Yao, Z.; Zhao, Y.; Wu, H. C.; Chem, A. Highly Sensitive Simultaneous Detection of Lead(II) and Barium(II) with G-quadruplex DNA in α-hemolysin Nanopore. Analytical chemistry 2013, 85 (15), 7302-7307. 29



(39) Zeng, T.; Li, T.; Li, Y.; Liu, L.; Wang, X.; Liu, Q.; Zhao, Y.; Wu, H. C. DNA-based Detection of Mercury(II) Ions through Characteristic Current Signals in Nanopores with High Sensitivity and Selectivity. Nanoscale 2014, 6 (15), 8579-8584. (40) Wen, S.; Zeng, T.; Liu, L.; Zhao, K.; Zhao, Y.; Liu, X.; Wu, H. C. Highly Sensitive and Selective DNA-based Detection of Mercury(II) with α-hemolysin Nanopore. Journal of the American Chemical Society 2011, 133 (45), 18312-18317. (41) Lin, X.; Ivanov, A. P.; Edel, J. B. Selective Single Molecule Nanopore Sensing of Proteins Using DNA Aptamer-Functionalised Gold Nanoparticles. Chemical Science 2017, 8 (5), 3905-3912. (42) Alibakhshi, M. A.; Halman, J. R.; Wilson, J.; Aksimentiev, A.; Afonin, K. A.; Wanunu, M. Picomolar Fingerprinting of Nucleic Acid Nanoparticles Using Solid-State Nanopores. Acs Nano 2017, 11 (10), 9701-9710. (43) Feng, Q. M.; Zhou, Z.; Li, M. X.; Zhao, W.; Xu, J. J.; Chen, H. Y. DNA Tetrahedral Scaffolds-based Platform for the Construction of Electrochemiluminescence Biosensor. Biosensors & Bioelectronics 2017, 90, 251-257. (44) Zhou, G.; Lin, M.; Song, P.; Chen, X.; Chao, J.; Wang, L.; Huang, Q.; Huang, W.; Fan, C.; Zuo, X. Multivalent Capture and Detection of Cancer Cells with DNA Nanostructured Biosensors and Multibranched Hybridization Chain Reaction Amplification. Analytical chemistry 2014, 86 (15), 7843-7848. (45) Zhi, G.; Lin, M.; Wang, P.; Pei H.; Juan Y.; Q, Huang.; D He.; Fan, C.; Zuo, X. Hybridization Chain Reaction Amplification of MicroRNA Detection with a Tetrahedral DNA Nanostructure-Based Electrochemical Biosensor. Analytical chemistry 2014, 86 (4), 2124-2130. (46) Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.; Zuo, X. Programmable Engineering of a Biosensing Interface with Tetrahedral DNA Nanostructures for Ultrasensitive DNA Detection. Angewandte Chemie International Edition 2015, 54 (7), 2151-2155. (47) Song, P.; Li, M.; Shen, J.; Pei, H.; Chao, J.; Su, S.; Aldalbahi, A.; Wang, L.; Shi, J.; Song, S. Dynamic Modulation of DNA Hybridization Using Allosteric DNA Tetrahedral Nanostructures. Analytical chemistry 2016, 88 (16), 8043-8049. (48) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA Nanostructures for Theranostic Applications. Accounts of chemical research 2013, 47 (2), 550-559. (49) Pei, H.; Lu, N.; Wen, Y.; Song, S.; Liu, Y.; Yan, H.; Fan, C. A DNA Nanostructure‐based Biomolecular Probe Carrier Platform for Electrochemical Biosensing. Advanced Materials 2010, 22 (42), 4754-4758. (50) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Reconfigurable Three‐dimensional DNA Nanostructures for the Construction of Intracellular Logic sensors. Angewandte Chemie International Edition 2012, 51 (36), 9020-9024. (51) Abi, A.; Lin, M.; Pei, H.; Fan, C.; Ferapontova, E. E.; Zuo, X. Electrochemical Switching with 3D DNA Tetrahedral Nanostructures Self-assembled at Gold Electrodes. ACS applied materials & interfaces 2014, 6 (11), 8928-8931. (52) Chen, Q.; Liu, H.; Lee, W.; Sun, Y.; Zhu, D.; Pei, H.; Fan, C.; Fan, X. Self-assembled DNA Tetrahedral Optofluidic Lasers with Precise and Tunable Gain Control. Lab on a chip 2013, 13 (17), 30


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3351-3354. (53) Qu, X.; Zhang, H.; Chen, H.; Aldalbahi, A.; Li, L.; Tian, Y.; Weitz, D. A.; Pei, H. Convection-Driven Pull-down Assays in Nanoliter Droplets Using Scaffolded Aptamers. Analytical chemistry 2017, 89 (6), 3468-3473. (54) Cui, H.; Xiong, X.; Gao, B.; Chen, Z.; Luo, Y.; He, F.; Deng, S.; Chen, L. A Novel Impedimetric Biosensor for Detection of Lead (II) with Low ‐ cost Interdigitated Electrodes Made on PCB. Electroanalysis 2016, 28 (9), 2000-2006. (55) Ali, M.; Tahir, M. N.; Siwy, Z.; Neumann, R.; Tremel, W.; Ensinger, W.; Chem, A. Hydrogen Peroxide Sensing with Horseradish Peroxidase-Modified Polymer Single Conical Nanochannels. Analytical chemistry 2011, 83 (5), 1673-1680. (56) Wanunu, M.; Dadosh, T.; Ray, V.; Jin, J.; Mcreynolds, L.; Drndić, M. Rapid Electronic Detection of Probe-Specific MicroRNAs Using Thin Nanopore Sensors. Nature nanotechnology 2010, 5 (11), 807-814. (57) Zhuang, J.; Fu, L.; Xu, M.; Zhou, Q.; Chen, G.; Tang, D. DNAzyme-based Magneto-Controlled Electronic Switch for Picomolar Detection of Lead (II) Coupling with DNA-based Hybridization Chain Reaction. Biosensors & Bioelectronics 2013, 45 (2), 52-57. (58) Pelossof, G.; Tel-Vered, R.; Willner, I. Amplified Surface Plasmon Resonance and Electrochemical Detection of Pb2+ Ions Using the Pb2+-Dependent DNAzyme and Hemin/G-Quadruplex as a Label. Analytical chemistry 2012, 84 (8), 3703-3709. (59) Heng, J. B.; Ho, C.; Kim, T.; Timp, R.; Aksimentiev, A.; Grinkova, Y. V.; Sligar, S.; Schulten, K.; Timp, G. Sizing DNA Using a Nanometer-Diameter Pore. Biophysical journal 2004, 87 (4), 2905-2911. (60) Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J.-F.; Dekker, C. Fast DNA Translocation through a Solid-State Nanopore. Nano letters 2005, 5 (7), 1193-1197. (61) Han, A.; Creus, M.; Schürmann, G.; Linder, V.; Ward, T. R.; de Rooij, N. F.; Staufer, U. Label-free Detection of Single Protein Molecules and Protein− Protein Interactions Using Synthetic Nanopores. Analytical chemistry 2008, 80 (12), 4651-4658. (62) Lan, W.-J.; Holden, D. A.; Zhang, B.; White, H. S. Nanoparticle Transport in Conical-Shaped Nanopores. Analytical chemistry 2011, 83 (10), 3840-3847.

Email: Libo Zhu: [email protected]; ORCID: 0000-0002-1986-3274 Ying Xu: [email protected] 31



Irshad Ali: [email protected] Liping Liu: [email protected] Hongwen Wu: [email protected] Zuhong Lu: [email protected] Quanjun Liu: [email protected]; ORCID: 0000-0002-9414-7892 TOC Graphic

32


Page 32 of 32

Solid-State Nanopore Single-Molecule Sensing of DNAzyme

Recommend Documents